1. Field of the Invention
The invention generally relates to methods to “fingerprint” or resolve the migration patterns of complex, polydisperse heparin mixtures. In particular, the invention provides methods for resolving complex heparins by adding polyamine resolving agents to the mixture prior to analysis of the heparins by a technique that separates macromolecules according to charge to mass ratio.
2. Background of the Invention
Anticoagulants are molecules used to treat and prevent a number of thrombotic disorders including pulmonary embolism, deep-vein thrombosis, disseminated intravascular coagulation, acute myocardial infarction, unstable angina, cerebrovascular thrombosis, and others. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US (1). Thus, a huge market exists for anticoagulants.
The two most commonly used anticoagulants are heparin (H) and low molecular weight heparin (LMWH). Heparin is a highly sulfated linear polysaccharide with an average molecular weight (MR) of ˜13,000 and is composed of alternating 1→4-linked uronic acid and glucosamine residues (FIG. 1). Clinically used heparin, appropriately named unfractionated heparin (UFH), is obtained from animal mucosa. UFH is a mixture of millions of chemical species differing from each other in size and chemical constitution (2,3).
LMW heparins are much smaller (MR˜5,000) and are produced by chemical or enzymatic depolymerization, or chromatographic separation of UFH (2). LMWHs also comprise of millions of distinct structures. In fact, they may contain additional non-native structures arising from the method of preparation.
Three LMWHs are currently approved by the Food and Drug Administration (FDA) including enoxaparin, tinzaparin, dalteparin. LMWHs are available in foreign markets such as Brazil, India, China, etc. and there is a growing discussion on the introduction of generic LMWHs in the US. LMWHs have gained market share since their introduction in 1993. The current market is approximately $4 billion per year in the US alone. Yet, they suffer from significant problems including enhanced bleeding risk, immunological reaction, patient-to-patient response variability, narrow therapeutic index, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio. These problems are elevated for UFH, while their frequency is reduced somewhat with LMWH.
The many adverse side effects of heparin and LMWH arise from their structure. The presence of large numbers of sulfate and carboxylate groups makes these polymers the strongest acid in human physiology. This acidity induces interaction with practically any protein that carries a cationic domain (4). A conservative estimate puts the number of heparin-binding proteins in the human body at more than 100. Yet, these interactions are different and unpredictable for different heparin or LMWH chains because their microscopic structures are different. These differences are perhaps the single major source of complications associated with heparin therapy. This structural heterogeneity has led the FDA to recommend that each LMWH should be considered as an independent drug with its own anticoagulant profile (5,6). Thus, patients on enoxaparin may not be routinely switched to tinzaparin and vice versa.
The differences in the structural and clinical profiles of LMWH have led to a large number of biophysical studies on developing methods for identifying these differences. Of particular interest is the ability to reliably assess product identity and quality in a routine manner, especially in view of the large number of sources of heparin and heparin-derived products that are available for medical use. Polyacrylamide gel electrophoresis in combination with cationic dye color development has been used to analyze heparin polydispersity (7-11). Likewise, size exclusion/gel permeation chromatography has been used to assess the average molecular weight and/or oligomeric composition of heparin (8,11-19). Both these techniques resolve UFH and LMWH into oligomers, especially the smaller chains, but do not provide more detailed structural information. On the other hand, chromatographic techniques, including reverse phase, ion-pairing and strong anion exchange, have been used to prepare heparin oligosaccharides as well as to perform oligosaccharide compositional analysis (20-27). More recently, a combination of liquid chromatography and mass spectrometry has been exploited to derive detailed sequence information on a variety of heparin preparations (28-33). Other techniques that have been utilized to understand UFH and LMWH structure and composition include capillary electrophoresis (CE) and nuclear magnetic resonance (NMR). NMR permits direct structure information from unmodified heparin chains and provides saccharide composition and sulfation pattern (34-37). It can also provide information on the average molecular weight of the heparin sample (18,38).
Capillary electrophoresis has been widely exploited to study UFH, LMWH and heparin oligosaccharides. The earliest application of CE to the analysis of heparins included disaccharide and oligosaccharide composition of UFH and LMWHs (39,40), which has now been modified to protocols with much better sensitivity and resolving power (41,42). Taking cue from the tandem HPLC—MS approaches to derive sequence information, a tandem CE—MS system has also been reported to perform disaccharide analysis (43). Unfortunately, these powerful systems work primarily on fragmented or smaller heparin oligomers. Analysis of unfragmented, intact LMWHs and unfractionated heparin is challenging because of the size of the biopolymers as well as its phenomenal charge. CE is a powerful technique that affords phenomenal resolution of nearly 100,000 theoretical plates, which is at least 10-fold higher than typical HPLC. CE relies on movement of charged particles and hence can be expected to be especially suited for the highly charged heparin chains. Polymeric natural and synthetic heparins have been assayed by CE techniques in low pH buffer using both the normal length (44) and short-end injection configuration (45). Typically, the main disadvantage of these polymeric species is that a wide peak is generally observed. The short end injection configuration enhanced efficiency, reduced analysis time and improved reproducibility. The analysis was highly sensitive to the pH of the buffer, but less so to the ionic strength. In an alternative approach, Toida and Linhardt have analyzed these polymers as copper complexes in an acidic buffer by reversed polarity (46), while Stefansson and Novotny have used cationic compounds to aid resolution (47). Since the introduction of CE analyses using reverse polarity (41), the trend has been to perform separations in either phosphate or formic acid buffers with pH in the range of 2 to 5. Under these acidic conditions, the Electroosmotic Force (EOF) is nearly eliminated and resolution is a direct function of the negative charge density and the structure of the analytes. Two groups have attempted to resolve LMWHs using these reverse polarity conditions with marginal results. Both Ramasamy et al. (48) and Patel et al. (49) have utilized a reverse polarity method in a bare fused silica capillary at pH 2.0-5.0 to resolve LMWH samples. Whereas the former utilized copper to detect heparin chains (240 nm), the latter utilized heparin's small absorbance at 230 nm. Yet, the resolution of LMWH samples was minimal. In fact, Patel et al. (49) observed a broad, heterogeneous peak with a width of several minutes, while Ramasamy et al. (48) reported small shoulders in the peak front portion of the electropherogram. These approaches are not useful to perform analysis of clinical LMWHs for assessing product identity and product quality. Thus, although several analytical methods are available, the prior art has thus far failed to provide an analytical method that can be implemented in routine manner to assess product identity and quality, and to authenticate the purported composition of heparin samples.